[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity:

negative

Based on following OECD conform studies:

- In Vitro Mammalian Chromosomal Aberration Test (according to OECD 473)

- In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes (according to OECD 476)

- Bacterial Reverse Mutation Test (according to OECD 471)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Untreated negative controls:

yes

Remarks:

Dimethyl sulphoxide

Positive controls:

yes

Remarks:

TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli

Details on test system and experimental conditions:

TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli

Rationale for test conditions:

The results of the study from both the initial and confirmatory mutation assay showed that, Licocare RBW 300 FL TP did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

Evaluation criteria:

Tester strain integrity
All Salmonella typhimurium tester strains and the Escherichia coli tester strain used in this assay must demonstrate their growth potential in the presence of histidine and tryptophan, respectively.

All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and ultraviolet light to demonstrate the presence of rfa mutation and uvrB mutation, respectively.

Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate the presence of uvrA mutation.

Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the plasmid R-factor.

• The spontaneous reversion rates in the vehicle control must be in the range of in-house historical data.
• There must be at least three non-toxic dose levels.
• The top dose selected should demonstrate toxicity. In case of non-toxic test items, the top dose tested should be 5000 µg/plate.
• All tester strain culture titers must be in the range of 1-2 x 109 cells/mL to ensure that appropriate numbers of bacteria are used for plating.
• The positive control substances should produce a more than 3-fold increase in mutant colony frequencies when compared to the respective vehicle control plates.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Results of Preliminary Toxicity Test

Treatment (mg/plate)

TA 100 revertant colonies/plate^*

Presence of Metabolic Activation

Absence of Metabolic Activation

Mean

Background lawn Intensity**

Precipitation**

Mean

Background lawn Intensity**

Precipitation**

DMSO

112

4+

NPO

113

4+

NPO

50

111

4+

NPO

107

4+

NPO

100

112

4+

NPO

107

4+

NPO

200

109

4+

NPO

108

4+

NPO

400

106

4+

NPO

107

4+

NPO

800

107

4+

NPO

105

4+

NPO

1600

108

4+

NPO

107

4+

NPO

3200

108

4+

NPO

107

4+

NPO

5000

106

4+

NPO

107

4+

NPO

*: Mean of two replicates                           **: Refer Appendix 7

               

Table 2. Viable Counts of the Overnight Culture of the Tester Strains

Tester Strains		Viable Counts (x 10^9CFU/mL*)
		Initial Mutation Assay		Confirmatory Mutation Assay
TA98		1.54		1.56
TA100		1.58		1.57
TA1535		1.55		1.56
TA1537		1.56		1.58
WP2uvrA (pKM101)		1.63		1.62

 

* Required Cell count: 1-2x10⁹Colony Forming Units (CFU)/mL

 

 

 

 

 

 

TALBE 3. Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	2	NA	108	3	NA	16	3	NA	13	3	NA	148	3	NA
50	25	2	0.96	108	2	1.00	16	2	1.02	13	2	1.00	146	3	0.99
158	26	2	1.00	105	2	0.97	15	2	0.94	13	2	0.95	147	4	0.99
500	26	2	1.01	105	3	0.98	14	2	0.88	12	1	0.90	146	4	0.99
1581	25	2	0.95	105	3	0.97	13	3	0.83	12	2	0.88	147	4	0.99
5000	25	3	0.95	106	3	0.98	13	2	0.83	11	1	0.83	146	2	0.99
Positive controls	364^c	8^c	14.01^c	765^c	15^c	7.08^c	163^c	6^c	10.21^c	164^c	9^c	12.33^c	576^d	8^d	3.89^d
^aValues are means of three replicates calculated from Appendix 3 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)                                               ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                                         SD: Standard deviation

 

 

 

 

 

 

Table 4. Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation

Treatment (µg/plate)	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA(pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	2	NA	107	2	NA	14	2	NA	14	2	NA	146	3	NA
50	24	3	0.95	107	3	1.00	15	2	1.07	12	3	0.90	148	2	1.01
158	25	2	0.97	107	5	1.00	15	2	1.02	12	2	0.90	148	4	1.02
500	25	2	0.97	104	3	0.98	14	3	0.98	13	2	0.93	145	3	0.99
1581	25	3	0.97	106	4	0.99	14	3	0.95	13	3	0.93	146	3	1.00
5000	24	2	0.93	105	4	0.98	14	2	1.00	13	2	0.93	144	3	0.99
Positive controls	273^c	9^c	10.65^c	562^d	7^d	5.26^d	152^d	10^d	10.63^d	163^e	12^e	11.92^e	566^f	14^f	3.89^f
^aValues are means of three replicates calculated from Appendix 4 and are rounded off to the nearest whole number  ^bRatio of treated/vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98: 2-Nitrofluorene (2 µg/plate),                     ^dTA100, TA1535: Sodium azide (1 µg/plate),       ^eTA1537: 9-Aminoacridine (50 µg/plate),             ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)                       NA: Not applicable                                                         SD: Standard deviation

 

 

 

 

 

 

Table 5. Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	26	3	NA	112	3	NA	17	2	NA	15	3	NA	147	5	NA
50	26	2	0.99	107	4	0.96	16	3	0.98	13	2	0.86	148	4	1.00
158	25	2	0.95	109	3	0.97	16	3	0.94	14	2	0.95	148	3	1.01
500	25	3	0.95	104	4	0.93	16	2	0.94	13	2	0.89	146	3	1.00
1581	26	2	1.00	107	3	0.96	15	3	0.88	13	2	0.86	146	4	1.00
5000	25	3	0.94	108	3	0.96	14	3	0.82	13	3	0.89	144	4	0.98
Positive controls	372^c	15^c	14.11^c	766^c	22^c	6.86^c	155^c	9^c	9.32^c	161^c	13^c	11.00^c	568^d	18^d	3.87^d
^aValues are means of three replicates calculated from Appendix 5 and are rounded off to the nearest whole number   ^bRatio of treated/vehicle control (mean revertants per plate).The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)               ^dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate) NA: Not applicable                                                                                                         SD: Standard deviation

 

 

 

 

 

 

 

Table 6. Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation

Treatment [µg/plate]	No. of revertants/plate^a
	TA98			TA100			TA1535			TA1537			WP2uvrA (pKM101)
	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b	Mean	SD	Ratio^b
DMSO	27	2	NA	108	2	NA	16	2	NA	14	3	NA	148	3	NA
50	25	2	0.95	107	4	0.99	15	2	0.92	12	2	0.85	147	3	0.99
158	27	2	1.00	106	3	0.98	15	1	0.90	12	1	0.85	146	4	0.99
500	25	3	0.94	108	3	1.00	16	3	1.00	11	2	0.78	146	4	0.99
1581	26	2	0.97	107	4	0.99	15	3	0.90	12	2	0.88	145	3	0.98
5000	25	2	0.94	103	3	0.96	14	2	0.88	12	2	0.90	145	4	0.98
Positive controls	276^c	15^c	10.36^c	567^d	19^d	5.25^d	158^d	11^d	9.65^d	166^e	11^e	12.12^e	564^f	8^f	3.80^f
^aValues are means of three replicates calculated from Appendix 6 and are rounded off to the nearest whole number ^bRatio of treated/vehicle control (mean revertants per plate). The presentation was made using the mean values with decimals before rounding off to the nearest whole number. Hence, some of the values may not match if calculated using the rounded-off mean values of this summary table. ^cTA98: 2-Nitrofluorene (2 µg/plate),                     ^dTA100, TA1535: Sodium azide (1 µg/plate)                         ^eTA1537: 9-Aminoacridine (50 µg/plate),              ^fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)                       NA: Not applicable                                                         SD: Standard deviation

 

 

 

Conclusions:

All criteria for a valid study were met as described in the study plan. It is concluded that the test item, Licocare RBW 300 FL TP, was not mutagenic in this bacterial reverse mutation test up to the OECD 471-recommended top dose of 5000 µg/plate under the conditions of testing employed.

Executive summary:

The test item, Licocare RBW 300 FL TP was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains ofSalmonella typhimuriumand WP2uvrA (pKM101) strain ofEscherichia coliin three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

Licocare RBW 300 FL TP formed a free flowing suspension in Dimethyl sulfoxide (DMSO) at 50 mg/mL.

 

In a preliminary toxicity test for the selection of test doses for the mutation assay, the test itemdid not precipitate on the basal agar plates at any of the tested doses.The test item did not show toxicity to the tester strain TA100 at any of the tested doses as the intensity of the bacterial background lawn as well as the mean number of revertant colonies were comparable to the vehicle control plates. Based on these observations, the test item was tested up to the OECD 471-recommended top dose of 5000 µg/plate in the mutation assay.

 

In the mutation assay, the bacterial tester strains were exposed to                       Licocare RBW 300 FL TPin triplicate at 50, 158, 500, 1581 and 5000mg/plate. The initial mutation assay was conducted using the direct plate incorporation mode of exposure and the confirmatory mutation assay was conducted using the pre-incubation mode of exposure. The vehicle control (DMSO) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

 

The results of the study from both the initial and confirmatory mutation assay showed that, Licocare RBW 300 FL TP did not show any positive mutagenic increase at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

 

The study indicated that Licocare RBW 300 FL TP was not mutagenic in this Bacterial Reverse Mutation Assay up to the OECD 471-recommended top dose of 5000mg/plate, under the conditions of testing employed.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September 2019 to 14 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Clariant Plastics and Coatings (Deutschland) GmbH

Target gene:

Hprt locus of CHO AA8 cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Alpha MEM
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate

Test concentrations with justification for top dose:

2 mg/mL as no change in pH and no precipitation was observed at and upto 2 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:As test item formed suspension in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 2×106 per flask (8×106 per group)

DURATION
- Exposure duration: 3 to 6 hours

NUMBER OF REPLICATIONS: 01

METHODS OF STAINING TECHNIQUE USED: Giemsa

Rationale for test conditions:

NA

Evaluation criteria:

Acceptance of test is based on the following criteria:
• Concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
• Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
• Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
• Adequate number of cells and concentrations are analyzable (according to OECD guidelines for testing of chemicals, No. 476).
• Criteria for selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=〖(X+A)〗^B
Where,
Y = transformed mutant frequency
X = observed mutant frequency
[Where X=(No. of mutant colonies per replicate)/(ACE value)×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

up to the highet dose tested ( 2 mg /ml)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Water solubility: Insoluble
- Precipitation: No

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Positive Control- Benzo(a)pyrene and 4- Nitroquinoline N-oxide
With Metabolic Activation
(3 to 6 hours)
[Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours)
[4 Nitroquinoline N-oxide]
Mean Data of Mutant Frequency/2x106 Cells 261.94 264.60
Standard
Deviation 27.28 18.52
Margin of Error 17.82 12.10
Upper bound 279.76 276.70
Lower bound 244.12 252.50

- solvent/vehicle historical control data:
Vehicle-DMSO
With Metabolic Activation
(3 to 6 hours) Without Metabolic Activation
(3 to 6 hours)
Mean Data of Mutant Frequency/2x106 Cells 24.51 25.43
Standard
Deviation 2.81 1.89
Margin of Error 1.95 1.31
Upper bound 26.46 26.74
Lower bound 22.56 24.12

Conclusions:

Based on the results obtained, the test item, LICOCARE RBW 300 FL TP is considered as non-mutagenic at and up to concentration of 2 mg/mL, both in presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test itemLICOCARE RBW 300 FL TPwas evaluated to identify gene mutations induced using genetic endpoints to measure mutation at hypoxanthine-guanine phosphoribosyl transferase (HPRT) using the gene mutation test in CHO AA8 cells.The test item formed suspension in dimethyl sulphoxide (DMSO) at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL.                       Post 3 hours of incubation, no change in pH and no precipitation was observed at and upto 2 mg/mL. On the basis of the results, 2 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.125, 0.25, 0.5, 1 and 2 mg/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours).The results of the initial cytotoxicity test indicated that the Relative Survival is greater  than 10 to 20 % at and upto 2 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on the initial cytotoxicity test results, 2 mg/mL was selected as highest concentration for gene mutation test. The gene mutation test was conducted at concentrations of 0.25, 0.5, 1 and 2 mg/mL using DMSO as a vehicle in four plates / group in the presence and absence of metabolic activation (3 to 6 hours).                                                                                                                                                                                                                                                                                                                          Benzo(a) pyrene and4 Nitroquinoline N-oxidewere used asPositive controlsfor the gene mutation test.Cytotoxicity was assessed by determining the Adjusted Cloning Efficiencies and Relative Survival in the test.There was no statistically significant increase in number of mutant frequencies at any of the concentrations tested when compared with the vehicle control.Positive controls resulted in mutant frequencies,which were statistically significant when compared with the vehicle control.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2019 to 19 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 473

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOLUBILTY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (15 to 25°C)
- Solubility of the test substance in the solvent/vehicle: Test item formed a suspension in dimethyl sulphoxide at 200 mg/mL

Species / strain / cell type:

lymphocytes:

Remarks:

Human blood

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral lymphocytes
- Suitability of cells: Healthy, young, non-smoking donors
- proliferation : 2% phytohaemagglutinin (PHA)
- Sex, age and number of blood donors if applicable: Human peripheral lymphocytes from the blood of healthy, young, non-smoking donors 30 and 29 years (male) of age with no known recent exposure to genotoxic chemicals or radiation were used.
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Methods for maintenance in cell culture if applicable: CO2 INCUBATOR
- Modal number of chromosomes:46±2

MEDIA USED
- Type and identity of media : RPMI-1640 Culture media
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 homogenate

Test concentrations with justification for top dose:

Test concentrations are 0.5, 1 and 2 mg/mL, as the Percent mitotic index (MI %) was not more than 45±5% at 2 mg/mL, same concentration was selected as highest concentration and the other concentrations selected were 0.5 and 1 mg/mL for the chromosomal aberration test

Vehicle / solvent:

Dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 to 6 hours and 20 to 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 3 to 6 hours and 20 to 24 hours



STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 02

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellets were mixed with 3 to 4 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 3 of cold acetic acid: methanol fixative (1:3).
Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried.
Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 minutes

NUMBER OF CELLS EVALUATED: 500 cells (Initial cytotoxicity)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 Metaphase per cultrue

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

Rationale for test conditions:

Not applicable

Evaluation criteria:

Considered acceptable for addition to the laboratory historical negative control database.
Adequate number of cells (at least 300 well spread metaphases per concentration) and concentrations (at least three analyzable concentrations) should be analyzed.

Statistics:

Yes

Key result

Species / strain:

lymphocytes:

Remarks:

human peripheral lymphocytes from blood

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 2 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

10 µg/mL of cyclophosphamide monohydrate in the presence of metabolic activation induced 10 % aberrant cells.

Key result

Species / strain:

lymphocytes:

Remarks:

uman peripheral lymphocytes from blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 2 mg/ mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No change in pH observed in any of the concentrations tested up to 2 mg/mL
- Water solubility: No
- Precipitation: Moderate and mild precipitation was observed at 2 and 1 mg/mL respectively and no precipitation observed in any other concentrations.

RANGE-FINDING/SCREENING STUDIES:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation
- Positive historical control data:

With S9 - cyclophosphamide monohydrate
(3 to 6 hours) Without S9 - Mitomycin C (3 to 6 hours) Without S9 - Mitomycin C (20 to 24 hours)
Average (% of Aberrated cells) 9.88 9.90 10.42
Standard Deviation 1.12 1.26 1.89
Sample size 16.00 16.00 16.00
Margin of error 0.55 0.62 0.93
Upper bound 10.43 10.52 11.34
Lower bound 9.33 9.28 9.49
95% Confidence level 1.96 1.96 1.96
Max 12.00 13.35 16.00
Min 8.67 8.67 8.67

- Negative (solvent/vehicle) historical control data:

With S9
(3-6 hours) Without S9 (3-6 hours) Without S9 (20-24 hours)
Average (% of Aberrated cells) 0.76 0.90 0.81
Standard Deviation 0.24 0.24 0.29
Sample size 12.00 12.00 12.00
Margin of error 0.14 0.14 0.16
Upper bound 0.90 1.04 0.97
Lower bound 0.62 0.76 0.65
95% Confidence level 1.96 1.96 1.96
Max 1.00 1.33 1.30
Min 0.35 0.67 0.34

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The percentage reduction in Mitotic Index
- Other observations when applicable: Metaphase counting

Remarks on result:

other: Non genotoxic

Remarks:

NON-CLASTOGENIC

Conclusions:

Summary:

Test item formed suspension in DMSO. Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 23 hours and 28 minutes of incubation, moderate and mild precipitation was observed at 2 and 1 mg/mL respectively and no precipitation observed in any other concentrations. No change in pH observed in any of the concentrations tested up to 2 mg/mL. Hence 2 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations tested were 0.125, 0.25, 0.5 and 1 mg/mL of test item.
The percentage reduction in Mitotic Index was in the range of 0.75 to 11.85 upto 2 mg/mL. As the percentage reduction in MI was not more than 45±5% at 2 mg/mL, same was selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.5 and 1 mg/mL.
In the chromosomal aberration test, the cells were treated with Licocare RBW 300 FL TP at the concentrations of 0.5, 1 and 2 mg/mL using DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mytomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.
The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, the cells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.
The observed mean percent aberrated cells at 0.5, 1 and 2 mg/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 1.33, 1.33, and 1.00 respectively. Similarly, the observed mean percent aberrated cells at 0.5, 1 and 2 mg/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 1.00, 1.67 and 1.34 respectively.
The observed mean percent aberrated cells at 0.5, 1 and 2 mg/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.00, 1.67 and 1.67 respectively.
The cultures treated with positive controls for the short-term period (3 to 6 hours) both in the presence and absence of metabolic activation, and for the long-term period (20 to 24 hours) in the absence of metabolic activation induced were 10.00%, 10.34% and 11.00% of aberrated cells respectively, which was statistically significant compared with the respective vehicle control. This demonstrated sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate.
The concurrent vehicle control values were within the 95% control limits of the distribution of the laboratory’s historical vehicle control database.

There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested.

Conclusion :

Based on the results obtained, the test item, Licocare RBW 300 FL TP is considered as non-clastogenic up to the concentration of 2 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

The test item,Licocare RBW 300 FL TPobtained fromClariant Plastics and Coatings (Deutschland) GMBH was evaluated for chromosomal aberrations in human lymphocytes, as per the OECD guideline for the testing of chemicals, No. 473 “In vitroMammalian Chromosomal AberrationTest” adopted on 29^thJuly 2016.

Based on the results obtained, the test item,Licocare RBW 300 FL TP isconsidered as non-clastogenic up to the concentration of 2 mg/mL both in the presence and absence of metabolic activation under the presented test conditions

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 December 2019 to 14 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Swiss

Remarks:

Albino

Details on species / strain selection:

Mouse is one of the recommended species by regulatory agencies for conducting in vivo Micronucleus test among rodents

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 18 to 23 g
- Assigned to test groups randomly: Through grouping and randomization
- Fasting period before study: NA
- Housing: Maximum of five animals of same sex and group were housed together in a standard polypropylene cage (L 280 × B 220 × H 140 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized paddy husk was used as a bedding material
- Diet (e.g. ad libitum): Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period
- Water (e.g. ad libitum):Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 to 22.9°C
- Humidity (%): 44 to 65%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12 hours dark and 12 hours light

IN-LIFE DATES: From: 26/12/2019 To: 05/03/2020

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Corn oil is universally accepted vehicle for acute oral dosing
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot no. (if required): A1712001

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Required quantity of test item was weighed as per the dose. The weighed test item was transferred to a clean mortar and ground using pestle by adding a small quantity of vehicle to get a uniform suspension. The content was transferred to a measuring cylinder. Again, a small quantity of Corn oil was added to the mortar rinsed and transferred to the measuring cylinder. The rinsing procedure was repeated until the test item was transferred completely into the measuring cylinder. The final volume was made up with corn oil to get the desired concentration as per the dose requirement. Formulation of the test item was prepared freshly every day before dosing

Duration of treatment / exposure:

The test item was administered through oral route once a day for 2 consecutive days using gavage cannula.

Frequency of treatment:

once a day for 2 consecutive days

Post exposure period:

18 to 24 hours

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Limit Study: 3 groups
10 (5 Males + 5 Females)

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s): As per inhouse validation study
- Route of administration: Oral
- Doses / concentrations: 100 mg/kg body weight/10 mg/mL

Tissues and cell types examined:

For each animal 500 erythrocytes (which include mature and immature erythrocytes) were scored to determine the Polychromatic Erythrocytes (PCE) and Normochromatic Erythrocytes (NCE) ratio, in order to determine PCE: total erythrocytes ratio.

For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In pre study, none of the doses produced observable toxic effects (no depression of bone marrow proliferation or other evidence of cytotoxicity were observed up to 2000 mg/kg) and hence; 2000 mg/kg was selected as the limit dose for both the sexes in Limit study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Post 18 to 24 hours (Day 3) of the second day treatment, all surviving animals were sacrificed by cervical dislocation, the femur was isolated from each animal for bone marrow collection

DETAILS OF SLIDE PREPARATION: Bone marrow cells were obtained by cut opening the epiphyses of femur bone immediately following sacrifice. The marrow was flushed out in to a centrifuge tube using the Fetal Bovine Serum (FBS). The femur bone marrow cells were centrifuged at about 2700 rpm for 10 minutes. Prior to smear preparation, the supernatant was discarded and the cell pellet is then resuspended in approximately 50 µL of Fetal Bovine Serum.
Minimum of three slides were prepared for pre study and main study per animal. Smears before staining was fixed by immersing the slides in methanol for approximately 5 to 6 minutes. The air dried slides were stained with May-Gruenwald and Giemsa stain for evaluation.




METHOD OF ANALYSIS:

OTHER:

Evaluation criteria:

In the dose range finding study for each animal, minimum of 500 erythrocytes (which include mature and immature erythrocytes) were scored to determine the Polychromatic Erythrocytes (PCE) and Normochromatic Erythrocytes (NCE) ratio, in order to determine PCE: total erythrocytes ratio. This result was used to determine the cytotoxicity of the test item.
In the limit study, all the slides including those of positive and vehicle controls were coded before microscopic evaluation to avoid group bias during evaluation.
For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCE: total erythrocytes ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs)

Statistics:

Body weight of both pre study and limit study was analyzed by SPSS, version No. 22 at a 95% level (p≤0.05) of significance. Inter group comparison of Body weight of Day 1, 2 and 3 were done. Slides from limit study were decoded after analysis; the number of PCE (Polychromatic erythrocytes), RBC (Red blood corpuscles), MNPCE (Micronucleated Polychromatic erythrocytes) and PCE/ total erythrocytes ratio (Polychromatic erythrocytes/ total erythrocytes) and frequency of MNPCE was calculated. The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95% level (p≤0.05) of significance. All analysis and comparisons were evaluated at the 95% level of confidence (p<0.05).
Statistically significant changes obtained were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (p<0.05)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Yes
- Solubility: Suspension in corn oil
- Clinical signs of toxicity in test animals: No clinical signs observed
- Evidence of cytotoxicity in tissue analyzed: No toxicity observed
- Harvest times: 18 to 24 hours of day 2 treatment

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Yes
- Ratio of PCE/NCE (for Micronucleus assay): Yes
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

INDIVIDUAL ANIMAL MICRONUCLEUS DATA - MAIN STUDY

Sex	Group & Dose (mg/kg)	Animal No.	Total NCEs	Total PCEs	PCE: Total Erythrocytes Ratio	Mean of PCE: Total Erythrocytes Ratio	SD	% Reduction Of PCE: Total Erythrocytes Ratio	Total No. of PCE's Scored	Total Number of MNPCEs	% of MNPCEs	Mean of % of MNPCEs
Male	G1 & 0	Mc5271	255	276	0.52	0.51	0.00	NA	4010	2	0.05	0.05
		Mc5272	254	266	0.51				4021	3	0.07
		Mc5273	253	267	0.51				4151	2	0.05
		Mc5274	256	266	0.51				4008	2	0.05
		Mc5275	255	270	0.51				4215	1	0.02
	G2 & 100 (CPA)	Mc5276	274	234	0.46	0.47*	0.01	7.84	4017	30	0.75	0.69*
		Mc5277	270	240	0.47				4032	26	0.64
		Mc5278	280	245	0.47				4011	25	0.62
		Mc5279	265	240	0.48				4017	30	0.75
		Mc5280	280	246	0.47				4015	27	0.67
	G3 & 2000	Mc5281	285	251	0.47	0.46*	0.01	9.80	4121	4	0.10	0.07
		Mc5282	270	231	0.46				4015	3	0.07
		Mc5283	275	225	0.45				4012	2	0.05
		Mc5284	286	244	0.46				4022	3	0.07
		Mc5285	280	240	0.46				4125	3	0.07

SD: Standard deviation, CPA: Cyclophosphamide Monohydrate, *: Statistically significant

INDIVIDUAL ANIMAL MICRONUCLEUS DATA - MAIN STUDY

Sex	Group & Dose (mg/kg)	Animal No.	Total NCEs	Total PCEs	PCE: Total erythrocytes ratio	Mean of PCE: Total erythrocytes ratio	SD	% Reduction of PCE: Total erythrocytes ratio	Total No. of PCE's scored	Total Number of MNPCEs	% of MNPCEs	Mean of % of MNPCEs
Female	G1 & 0	Mc5286	260	266	0.51	0.50	0.01	NA	4125	2	0.05	0.05
		Mc5287	265	264	0.50				4085	1	0.02
		Mc5288	255	258	0.50				4151	2	0.05
		Mc5289	258	263	0.50				4063	3	0.07
		Mc5290	265	258	0.49				4017	2	0.05
	G2 & 100 (CPA)	Mc5291	290	243	0.46	0.47*	0.01	6.00	4021	25	0.62	0.65*
		Mc5292	287	259	0.47				4125	30	0.73
		Mc5293	279	248	0.47				4063	27	0.66
		Mc5294	280	266	0.49				4011	25	0.62
		Mc5295	289	257	0.47				4032	25	0.62
	G3 & 2000	Mc5296	270	230	0.46	0.45*	0.01	10.00	4015	2	0.10	0.08
		Mc5297	275	235	0.46				4101	3	0.07
		Mc5298	278	223	0.45				4022	4	0.10
		Mc5299	288	223	0.44				4021	3	0.07
		Mc5300	287	248	0.46				4031	2	0.05

SD: Standard deviation, CPA: Cyclophosphamide Monohydrate, *: Statistically significant

Conclusions:

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, Licocare RBW 300 FL TP is non-genotoxic at the limit dose of 2000 mg/kg.

Executive summary:

Test item,Licocare RBW 300 FLwas evaluated in the MammalianErythrocyte Micronucleus Test.

This study was conducted to determine the genotoxic potential ofLicocare RBW 300 FL TP inthe micronucleus test using bone marrow cells of Swiss Albino mice. The pre study consisted of four groups, vehicle control, 500, 1000 and 2000 mg/kg.Licocare RBW 300 FL TPwasadministered at a dose volume of 10 mL/kg. In the pre study, each group of mice consisted of 3 males and 3 females. The main study consisted of 3 groups of mice and each group consisted of 5 males and 5 females. G1 group animals were administered with vehicle, G2 group animals were administered with cyclophosphamide monohydrate at 100 mg/kg and animals of G3 group were administered with a limit dose of 2000 mg/kg for two consecutive days by oral route using gavage cannula. Post 18 to 24 hours of last dosing, all mice were sacrificed by cervical dislocation and bone marrow cells were collected. The slides of bone marrow cells were stained with May-Gruenwald and Giemsa stain and observed for incidences of micronucleated polychromatic erythrocytes (MNPCE).

In pre study, no clinical signs,no body weight changes, no gross pathological findingsand no mortality were observed in any of the animals dosed withLicocare RBW 300 FL TPin any of the dose levels.

In pre study, there was statistical significant decrease in thePCE: total erythrocytesratio at 500, 1000 and 2000 mg/kg in females and in males at 2000 mg/kgwhen compared to vehicle control group. The reduction in PCE: total erythrocytes was well below 50% (3.92% in males and 9.80% in females) and the test item was therefore considered not to be cytotoxic to the bone marrow. In pre study, none of the doses produced observable toxic effects (no depression of bone marrow proliferation or other evidence of cytotoxicity) hence,2000 mg/kg was selected as the limit dose for both the sexes in main study.

In the main study, there was no clinical signs, no body weight changes, no gross pathological findings and no mortality were observed in any of the animals dosed with test item at 2000 mg/kg and also in positive control dosed animals. There was statistical significant decrease in proportions of PCE: total erythrocytes ratio in animals dosed at 2000 mg/kg of test item and also in positive control group in both the sexes when compared to vehicle control group. The average percentage of MNPCEs was 0.05 in both the sexes dosed with vehicle. There was no statistically significant increase in thepercentageof micronucleated PCEs (per 4000 PCEs scored) at 2000 mg/kg in comparison with vehicle control.

The positive control,cyclophosphamide monohydrate at 100 mg/kgexhibited statistically significant increase in the percentage of MNPCE when compared to vehicle control. Thisdemonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were adequate.

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro/in vivo data are regarded as relevant for humans.

Additional information

Due to the findings in the performed Ames test no classification for mutagenicity is recommended according to the criteria of Regulation (EC) No 1272/2008.